Search

The functionalization of bone substitutes with exosomes appears to be a promising technique to enhance bone tissue formation. This study investigates the potential of exosomes derived from bone marrow mesenchymal stromal cells (BMSCs) to improve bone healing and bone augmentation when incorporated into wide open-porous 3D-printed ceramic Gyroid scaffolds. We demonstrated the multipotent characteristics of BMSCs and characterized the extracted exosomes using nanoparticle tracking analysis and proteomic profiling. Through cell culture experimentation, we demonstrated that BMSC-derived exosomes possess the ability to attract cells and significantly facilitate their differentiation into the osteogenic lineage. Furthermore, we observed that scaffold architecture influences exosome release kinetics, with Gyroid scaffolds exhibiting slower release rates compared to Lattice scaffolds. Nevertheless, in vivo implantation did not show increased bone ingrowth in scaffolds loaded with exosomes, suggesting that the scaffold microarchitecture and material were already optimized for osteoconduction and bone augmentation. These findings highlight the lack of understanding about the optimal delivery of exosomes for osteoconduction and bone augmentation by advanced ceramic scaffolds.

Triply periodic minimal surface microarchitectures (TPMS) were developed by mathematicians and evolved in all kingdoms of living organisms. Renowned for their lightweight yet robust attributes, TPMS structures find application in diverse fields, such as the construction of satellites, aircrafts, and electric vehicles. Moreover, these microarchitectures, despite their intricate geometric patterns, demonstrate potential for application as bone substitutes, despite the inherent gothic style of natural bone microarchitecture. Here, we produced three TPMS microarchitectures, D-diamond, G-gyroid, and P-primitive, by 3D printing from hydroxyapatite. We explored their mechanical characterization and, further, implanted them to study their bone augmentation and osteoconduction potential. In terms of strength, the D-diamond and G-gyroid performed significantly better than the P-primitive. In a calvarial defect model and a calvarial bone augmentation model, where osteoconduction is determined as the extent of bony bridging of the defect and bone augmentation as the maximal vertical bone ingrowth, the G-gyroid performed significantly better than the P-primitive. No significant difference in performance was observed between the G-gyroid and D-diamond. Since, in real life, the treatment of bone deficiencies in patients comprises elements of defect bridging and bone augmentation, ceramic scaffolds with D-diamond and G-gyroid microarchitectures appear as the best choice for a TPMS-based scaffold in bone tissue engineering.

The early phase of bone healing is a complex and poorly understood process. With additive manufacturing, we can generate a specific and customizable library of bone substitutes to explore this phase. In this study, we produced tricalcium phosphate-based scaffolds with microarchitectures composed of filaments of 0.50 mm in diameter, named Fil050G, and 1.25 mm named Fil125G, respectively. The implants were removed after only 10 days in vivo followed by RNA sequencing (RNAseq) and histological analysis. RNAseq results revealed upregulation of adaptive immune response, regulation of cell adhesion, and cell migration-related genes in both of our two constructs. However, significant overexpression of genes linked to angiogenesis, regulation of cell differentiation, ossification, and bone development was observed solely in Fil050G scaffolds. Moreover, quantitative immunohistochemistry of structures positive for laminin revealed a significantly higher number of blood vessels in Fil050G samples. Furthermore, µCT detected a higher amount of mineralized tissue in Fil050G samples suggesting a superior osteoconductive potential. Hence, different filament diameters and distances in bone substitutes significantly influence angiogenesis and regulation of cell differentiation involved in the early phase of bone regeneration, which precedes osteoconductivity and bony bridging seen in later phases and as consequence, impacts the overall clinical outcome.

Revolutionary fabrication technologies such as three-dimensional (3D) printing to develop dental structures are expected to replace traditional methods due to their ability to establish constructs with the required mechanical properties and detailed structures. Three-dimensional printing, as an additive manufacturing approach, has the potential to rapidly fabricate complex dental prostheses by employing a bottom-up strategy in a layer-by-layer fashion. This new technology allows dentists to extend their degree of freedom in selecting, creating, and performing the required treatments. Three-dimensional printing has been narrowly employed in the fabrication of various kinds of prostheses and implants. There is still an on-demand production procedure that offers a reasonable method with superior efficiency to engineer multifaceted dental constructs. This review article aims to cover the most recent applications of 3D printing techniques in the manufacturing of dental prosthetics. More specifically, after describing various 3D printing techniques and their advantages/disadvantages, the applications of 3D printing in dental prostheses are elaborated in various examples in the literature. Different 3D printing techniques have the capability to use different materials, including thermoplastic polymers, ceramics, and metals with distinctive suitability for dental applications, which are discussed in this article. The relevant limitations and challenges that currently limit the efficacy of 3D printing in this field are also reviewed. This review article has employed five major scientific databases, including Google Scholar, PubMed, ScienceDirect, Web of Science, and Scopus, with appropriate keywords to find the most relevant literature in the subject of dental prostheses 3D printing.

Tendon injuries can result in two major drawbacks. Adhesions to the surrounding tissue may limit the range of motion, while fibrovascular scar formation can lead to poor biomechanical outcomes. Prosthetic devices may help to mitigate those problems. Emulsion electrospinning was used to develop a novel three-layer tube based on the polymer DegraPol (DP), with incorporated insulin-like growth factor-1 (IGF-1) in the middle layer. Scanning electron microscopy was utilized to assess the fiber diameter in IGF-1 containing pure DP meshes. Further characterization was performed with Fourier Transformed Infrared Spectroscopy, Differential Scanning Calorimetry, and water contact angle, as well as through the assessment of mechanical properties and release kinetics from ELISA, and the bioactivity of IGF-1 by qPCR of collagen I, ki67, and tenomodulin in rabbit Achilles tenocytes. The IGF-1-containing tubes exhibited a sustained release of the growth factor up to 4 days and showed bioactivity by significantly upregulated ki67 and tenomodulin gene expression. Moreover, they proved to be mechanically superior to pure DP tubes (significantly higher fracture strain, failure stress, and elastic modulus). The novel three-layer tubes intended to be applied over conventionally sutured tendons after a rupture may help accelerate the healing process. The release of IGF-1 stimulates proliferation and matrix synthesis of cells at the repair site. In addition, adhesion formation to surrounding tissue can be reduced due to the physical barrier.

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals.

Autologous bone remains the gold standard bone substitute in clinical practice. Therefore, the microarchitecture of newly developed synthetic bone substitutes, which reflects the spatial distribution of materials in the scaffold, aims to recapitulate the natural bone microarchitecture. However, the natural bone microarchitecture is optimized to obtain a mechanically stable, lightweight structure adapted to the biomechanical loading situation. In the context of synthetic bone substitutes, the application of a Triply Periodic Minimum Surface (TPMS) algorithm can yield stable lightweight microarchitectures that, despite their demanding architectural complexity, can be produced by additive manufacturing. In this study, we applied the TPMS derivative Adaptive Density Minimal Surfaces (ADMS) algorithm to produce scaffolds from hydroxyapatite (HA) using a lithography-based layer-by-layer methodology and compared them with an established highly osteoconductive lattice microarchitecture. We characterized them for compression strength, osteoconductivity, and bone regeneration. The in vivo results, based on a rabbit calvaria defect model, showed that bony ingrowth into ADMS constructs as a measure of osteoconduction depended on minimal constriction as it limited the maximum apparent pore diameter in these scaffolds to 1.53 mm. Osteoconduction decreased significantly at a diameter of 1.76 mm. The most suitable ADMS microarchitecture was as osteoconductive as a highly osteoconductive orthogonal lattice microarchitecture in noncritical- and critical-size calvarial defects. However, the compression strength and microarchitectural integrity in vivo were significantly higher for scaffolds with their microarchitecture based on the ADMS algorithm when compared with high-connectivity lattice microarchitectures. Therefore, bone substitutes with high osteoconductivity can be designed with the advantages of the ADMS-based microarchitectures. As TPMS and ADMS microarchitectures are true li

Additive manufacturing enables the realization of the macro- and microarchitecture of bone substitutes. The macroarchitecture is determined by the bone defect and its shape makes the implant patient specific. The preset distribution of the 3D-printed material in the macroarchitecture defines the microarchitecture. At the lower scale, the nanoarchitecture of 3D-printed scaffolds is dependent on the post-processing methodology such as the sintering temperature. However, the role of microarchitecture and nanoarchitecture of scaffolds for osteoconduction is still elusive. To address these aspects in more detail, we produced lithography-based osteoconductive scaffolds from hydroxyapatite (HA) of identical macro- and microarchitecture and varied their nanoarchitecture, such as microporosity, by increasing the maximum sintering temperatures from 1100 to 1400 °C. The different scaffold types were characterized for microporosity, compression strength, and nanoarchitecture. The in vivo results, based on a rabbit calvarial defect model showed that bony ingrowth, as a measure of osteoconduction, was independent from scaffold's microporosity. The same applies to in vitro osteoclastic resorbability, since on all tested scaffold types, osteoclasts formed on their surfaces and resorption pits upon exposure to mature osteoclasts were visible. Thus, for wide-open porous HA-based scaffolds, a low degree of microporosity and high mechanical strength yield optimal osteoconduction and creeping substitution. Based on our study, non-unions, the major complication during demanding bone regeneration procedures, could be prevented.

Additive manufacturing can be applied to produce personalized bone substitutes. At present, the major three-dimensional (3D) printing methodology relies on fila­ment extrusion. In bioprinting, the extruded filament consists mainly of hydrogels, in which growth factors and cells are embedded. In this study, we used a lithogra­phy-based 3D printing methodology to mimic filament-based microarchitectures by varying the filament dimension and the distance between the filaments. In the first set of scaffolds, all filaments were aligned toward bone ingrowth direction. In a second set of scaffolds, which were derived from the identical microarchitecture but tilted by 90°, only 50% of the filaments were in line with the bone ingrowth direction. Testing of all tricalcium phosphate-based constructs for osteoconduction and bone regeneration was performed in a rabbit calvarial defect model. The results revealed that if all filaments are in line with the direction of bone ingrowth, filament size and distance (0.40–1.25 mm) had no significant influence on defect bridging. Howev­er, with 50% of filaments aligned, osteoconductivity declined significantly with an increase in filament dimension and distance. Therefore, for filament-based 3D- or bio-printed bone substitutes, the distance between the filaments should be 0.40 to 0.50 mm irrespective of the direction of bone ingrowth or up to 0.83 mm if perfectly aligned to it.

The human skeleton is a dynamic and remarkably organized organ system that provides mechanical support and performs a variety of additional functions. Bone tissue undergoes constant remodeling; an essential process to adapt architecture/resistance to growth and mechanical needs, but also to repair fractures and micro-damages. Despite bone's ability to heal spontaneously, certain situations require an additional stimulation of bone regeneration, such as non-union fractures or after tumor resection. Among the growth factors used to increase bone regeneration, bone morphogenetic protein-2 (BMP2) is certainly the best described and studied. If clinically used in high quantities, BMP2 is associated with various adverse events, including fibrosis, overshooting bone formation, induction of inflammation and swelling. In previous studies, we have shown that it was possible to reduce BMP2 doses significantly, by increasing the response and sensitivity to it with small molecules called "BMP2 enhancers". In the present study, we investigated the effect of N-Vinyl-2-pyrrolidone (NVP) on osteoblast and osteoclast differentiation in vitro and guided bone regeneration in vivo. We showed that NVP increases BMP2-induced osteoblast differentiation and decreases RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, in a rabbit calvarial defect model, the histomorphometric analysis revealed that bony bridging and bony regenerated area achieved with NVP-loaded poly (lactic-co-glycolic acid (PLGA) membranes were significantly higher compared to unloaded membranes. Taken together, our results suggest that NVP sensitizes BMP2-dependent pathways, enhances BMP2 effect, and inhibits osteoclast differentiation. Thus, NVP could prove useful as "osteopromotive substance" in situations where a high rate of bone regeneration is required, and in the management of bone diseases associated with excessive bone resorption, like osteoporosis.

N-methyl pyrrolidone (NMP) is an FDA approved molecule used as an excipient in pharmaceutical industry. Besides having a central role in formulation of drugs, the most important function of any excipient is to guarantee the safety of the medicine during and after its administration. Several studies have shown that exposure to NMP and especially in rats produce a gonadotoxic effect leading to infertility. However, the mechanisms underlying the effect of NMP on male reproduction are unknown. The aim of this study was to assess the reproductive toxicity of NMP in male rats and to elucidate the underlying mechanism. Male Sprague Dawley rats were injected intraperitoneally, twice/ week, at a dose of 108 mg/ 100 g of body weight with NMP. Analysis of reproductive parameters revealed testicular atrophy in NMP treated animals compared to control animals. Germ cell composition within the seminiferous tubules was disturbed and manifested in an increase in number of cells with fragmented DNA. A subsequent decrease in number of spermatocytes and spermatids was observed. Alpha screen assay shows that NMP acts at the concentrations we applied in vivo as a low affinity inhibitor for BRDT (testis specific bromodomain protein). BRDT inhibition is mirrored by a significant decrease in the expression of early stage spermatocyte markers (lmna, aurkc and ccna1), during which BRDT expression predominates. A significant decrease in testosterone levels was also observed. Since NMP interferes with spermatogenesis on various levels, its use in humans must be carefully monitored.

The treatment of bone defects with recombinant bone morphogenetic protein-2 (BMP-2) requires high doses precluding broad clinical application. Here, a bioengineering approach is presented that strongly improves low-dose BMP-2-based bone regeneration by mobilizing healing-associated mesenchymal progenitor cells (MPCs). Smart synthetic hydrogels are used to trap and study endogenous MPCs trafficking to bone defects. Hydrogel-trapped and prospectively isolated MPCs differentiate into multiple lineages in vitro and form bone in vivo. In vitro screenings reveal that platelet-derived growth factor BB (PDGF-BB) strongly recruits prospective MPCs making it a promising candidate for the engineering of hydrogels that enrich endogenous MPCs in vivo. However, PDGF-BB inhibits BMP-2-mediated osteogenesis both in vitro and in vivo. In contrast, smart two-way dynamic release hydrogels with fast-release of PDGF-BB and sustained delivery of BMP-2 beneficially promote the healing of bone defects. Collectively, it is shown that modulating the dynamics of endogenous progenitor cells in vivo by smart synthetic hydrogels significantly improves bone healing and holds great potential for other advanced applications in regenerative medicine.

The treatment of bone defects with recombinant bone morphogenetic protein-2 (BMP-2) requires high doses precluding broad clinical application. Here, a bioengineering approach is presented that strongly improves low-dose BMP-2-based bone regeneration by mobilizing healing-associated mesenchymal progenitor cells (MPCs). Smart synthetic hydrogels are used to trap and study endogenous MPCs trafficking to bone defects. Hydrogel-trapped and prospectively isolated MPCs differentiate into multiple lineages in vitro and form bone in vivo. In vitro screenings reveal that platelet-derived growth factor BB (PDGF-BB) strongly recruits prospective MPCs making it a promising candidate for the engineering of hydrogels that enrich endogenous MPCs in vivo. However, PDGF-BB inhibits BMP-2-mediated osteogenesis both in vitro and in vivo. In contrast, smart two-way dynamic release hydrogels with fast-release of PDGF-BB and sustained delivery of BMP-2 beneficially promote the healing of bone defects. Collectively, it is shown that modulating the dynamics of endogenous progenitor cells in vivo by smart synthetic hydrogels significantly improves bone healing and holds great potential for other advanced applications in regenerative medicine.

Additive manufacturing is a key technology required to realize the production of a personalized bone substitute that exactly meets a patient's need and fills a patient-specific bone defect. Additive manufacturing can optimize the inner architecture of the scaffold for osteoconduction, allowing fast and reliable defect bridging by promoting rapid growth of new bone tissue into the scaffold. The role of scaffold microporosity/nanoarchitecture in osteoconduction remains elusive. To elucidate this relationship, we produced lithography-based osteoconductive scaffolds from tricalcium phosphate (TCP) with identical macro- and microarchitecture, but varied their nanoarchitecture/microporosity by ranging maximum sintering temperatures from 1000 °C to 1200 °C. After characterization of the different scaffolds' microporosity, compression strength, and nanoarchitecture, we performed in vivo studies that showed that ingrowth of bone as an indicator of osteoconduction significantly decreased with decreasing microporosity. Moreover, at the 1200 °C peak sinter temperature and lowest microporosity, osteoclastic degradation of the material was inhibited. Thus, even for wide-open porous TCP-based scaffolds, a high degree of microporosity appears to be essential for optimal osteoconduction and creeping substitution, which can prevent non-unions, the major complication during bone regeneration procedures.

N,N-Dimethylacetamide (DMA) is FDA approved as an excipient and is used as drug-delivery vehicle. Due to its amphipathic nature and diverse bioactivities, it appears to be a good combination of biodegradable poly-lactide-co-glycolide (PLGA)-based guided bone regeneration membranes. Here we show that the solvent DMA can be loaded to PLGA membranes by different regimes, leading to distinct release profiles, and enhancing the bone regeneration in vivo. Our results highlight the potential therapeutic benefits of DMA in guided bone regeneration procedures, in combination with biodegradable PLGA membranes.

From the beginning of 2020, the governments and the health systems around the world are tackling infections and fatalities caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) resulting in the coronavirus disease 2019 (COVID-19). This virus pandemic has turned more complicated as individuals with co-morbidities like diabetes, cardiovascular conditions and obesity are at a high risk of acquiring infection and suffering from a more severe course of disease. Prolonged viral infection and obesity are independently known to lower the immune response and a combination can thus result in a "cytokine storm" and a substantial weakening of the immune system. With the rise in obesity cases globally, the chances that obese individuals will acquire infection and need hospitalization are heightened. In this review, we discuss why obesity, a low-grade chronic inflammation, contributes toward the increased severity in COVID-19 patients. We suggest that increased inflammation, activation of renin-angiotensin-aldosterone system, elevated adipokines and higher ectopic fat may be the factors contributing to the disease severity, in particular deteriorating the cardiovascular and lung function, in obese individuals. We look at the many lessons learnt from the 2009 H1N1 influenza A pandemic and relate it to the very little but fast incoming information that is available from the SARS-CoV-2 infected individuals with overweight and obesity. Keywords: COVID-19; SARS-CoV-2; adipocytes; adiposity; novel corona virus; obesity.

Development of an effective male contraceptive agent remains a challenge. The present study evaluates the potential of N, N-Dimethylacetamide (DMA), a FDA approved excipient as a male contraceptive agent. Male Sprague Dawley rats injected with DMA for a period of 8 weeks (one injection per week) showed a significant alteration of reproductive parameters. Furthermore, DMA treated animals showed complete infertility in a dose dependent manner, as no pups were born despite proper mating between females and DMA treated males. However, stopping the DMA treatment for a period of 8 weeks (after the initial treatment) restored the reproductive parameters to normal. Moreover, the fertility was resumed to normal as pups were born in the groups where DMA treatment was halted after initial DMA treatment. All these changes had no effect on the level of reproductive hormones FSH, LH and testosterone. Taken together, our results indicate that DMA acts in a reversible and non-hormonal manner to achieve contraception in rats. Therefore, repurposing the use of DMA could lead in a short time to an inexpensive and safer male contraceptive option. Keywords: BET inhibitor; bromodomain (BRD); excipient; male contraception; rat; reproduction.

Regenerative endodontics has been described as a paradigm shift in dentistry, despite its current limitation to immature teeth and reparative rather than regenerative outcomes. Cell-free treatments are favored because of regulatory issues. However, the recruitment of host-derived stem cells to the desired site remains challenging. We investigated whether dental pulp-derived exosomes, which are extracellular vesicles that contain proteins, lipids, RNA, and DNA and thus mirror their parental cells, may be used for this purpose. The use of exosomes may present appreciable advantages over the direct use of transplanted stem cells due to a higher safety profile, easier isolation, preservation, and handling. Here we harvested exosomes from a cultured third-molar pulp cell and assessed them by transmission electron microscopy and Western blotting. Human mesenchymal stem cells (MSCs) were exposed to these exosomes to assess exosome uptake, cell migration, and proliferation. In addition, a fibrin gel (i.e., a diluted fibrin sealant), was assessed as a delivery system for the exosomes. Our results show that exosomes attracted MSCs, and the fibrin gel enhanced their effect. Moreover, exosomes improved the proliferation of MSCs. Therefore, we propose that pulp-derived exosomes in combination with a fibrin gel could be a powerful combination for clinical translation towards improved cell-free regenerative endodontics and thus represent a new way to fill dental hard tissues.